The objectives of this project are to (1) use recombinant DNA techniques to express specific antigens of Borrelia burgdorferi to improve the serodiagnosis of Lyme disease, (2) characterize at the molecular level, isolates of the Lyme disease spirochete from a wide range of biological and geographical sources, and (3) identify molecular determinants of the spirochete that are important for infectivity in mammalian and tick hosts. As part of our continued interest in the 39 kilodalton (P39) protein of B. burgdorferi as a diagnostic antigen, analyses were completed with other species of bacteria. Five serovars of Leptospira interrogans, L. biflexa, Leptonema illini, and Rickettsia rickettsii were examined and found not to contain this antigen. The specificity of this antigen and its reactivity with human Lyme disease sera should exclude the possibility of false-positive serum samples from patients having had either leptospirosis or Rocky Mountain spotted fever, as well as tick-borne relapsing fever and syphilis, as we discussed in previous reports. We have examined 31 isolates of B. burgdorferi from pools of Ixodes pacificus ticks collected from numerous regions spanning most of the state of California where this tick is found. All isolates were identified as B. burgdorferi by their reactivities with monoclonal antibodies to outer surface proteins A and B, flagellin, and the 39 kDa (P39) protein. By polymerase chain reaction (PCR) assays, all of the isolates reacted as North American-type B. burgdorferi. Plasmid profiles were quite diverse and only lo of 27 isolates examined contained the small 8 kilobase supercoiled plasmid we described previously. One isolate was made from ticks collected at Wawona Campground in Yosemite National Park, documenting the occurrence of the Lyme spirochete in an area of extreme human recreational use. We have also developed a rapid technique to identify small supercoiled plasmids by photonicking the supercoiled molecules with UV light and ethidium bromide in agarose gels. This allows us to then identify newly-formed open circular molecules that migrate slower than supercoiled molecules in the gel.